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PubMed2003

Ectomycorrhizal fungal species and strains differ in their ability to produce free and conjugated polyamines.

Production of free and conjugated polyamines by one strain of Laccaria proxima (Boud.) Maire, three strains (H, O, K) of Paxillus involutus (Batsch) Fr., and one strain of Pisolithus tinctorius was studied in vitro. Spermidine (Spd) was the main polyamine in the 4-week-old mycelium of all the fungi. It was mainly present in the free form, but it also occurred in conjugated forms. Paxillus involutus strain H released large amounts of free putrescine (Put), and the Pisolithus tinctorius released a compound probably related to cadaverine (Cad). On the other hand, these two fungi contained less conjugated polyamines than the other fungi. In addition to the amounts, the forms (perchloric acid soluble and insoluble) of conjugated polyamines in the mycelium varied between species and strains. L. proxima contained nearly as much insoluble conjugated Spd as free Spd, whereas Paxillus involutus strains O and K contained relatively large amounts of soluble conjugated Spd. The results suggest that ectomycorrhizal fungal species and strains differ in their ability and need to produce conjugated polyamines. The small amounts of soluble conjugated polyamines found in the culture filtrates indicate that some specific conjugated polyamines may be involved in polyamine translocation across the plasma membrane.

Agaricales↗
PubMed1978

Determination of acetaldehyde in rat blood by the use of rat liver aldehyde dehydrogenase.

A method has been developed for the determination of low concentrations of acetaldehyde in rat blood. The method involves extraction of blood in perchloric acid followed by a fluorimetric determination of acetaldehyde in neutralized extracts by the use of a low K(m) aldehyde dehydrogenase isolated from rat liver mitochondria. Acetaldehyde concentrations down to 2 to 3 microM could be detected in blood samples of 0.1 ml containing high concentrations of ethanol (10-40 mM). Due to its simplicity, sensitivity, and the use of a low-cost fluorimeter, this enzymatic method should be a valuable complement to gas chromatographic methods for acetaldehyde determination.

Acetaldehyde↗
PubMed1983

The analysis of acyl-coenzyme A derivatives by reverse-phase high-performance liquid chromatography.

A method for determining tissue levels of Coenzyme A and various short-chain-length acyl-CoA derivatives using high-performance liquid chromatography is presented. Separation of the various compounds was accomplished using a reverse-phase Spherisorb ODS II, 5-microns C18 column. Mobile-phase solvents were (a) potassium phosphate, 220 mM; thiodiglycol (2,2-thiodiethanol), 0.05% (v/v), pH 4.0 and (b) methanol, 98%; chloroform; 2% (v/v). The various acyl-CoA derivatives were detected by monitoring the column effluent at 254 nm. Nearly baseline separation was obtained for a standard mixture of free CoASH, methylmalonyl-CoA, beta-hydroxy-beta-methylglutaryl-CoA, succinyl-CoA, acetoacetyl-CoA, acetyl-CoA, propionyl-CoA, isobutyryl-CoA, beta-methyl-crotonyl-CoA, and isovaleryl-CoA. CoA derivative profiles were determined in neutralized perchloric acid extracts of perfused rat hearts and livers and of isolated rat liver mitochondria to demonstrate the utility of this method for assessing the levels of CoA derivatives in biological samples.

Acyl Coenzyme A↗
PubMed1983

A spectrophotometric, enzymatic assay for D-3-hydroxybutyrate that is not dependent on hydrazine.

Because of the potential carcinogenic properties of hydrazine and because of other health hazards associated with its use in the laboratory, an enzymatic assay has been developed for D-3-hydroxybutyrate that is not dependent on hydrazine to drive the reaction toward completion. The use of a high concentration of NAD+ and a buffer at pH 9.5 resulted in a favorable conversion of D-3-hydroxybutyrate to acetoacetate by D-3-hydroxybutyrate dehydrogenase even though the reaction favors D-3-hydroxybutyrate formation under physiological conditions. The assay was also completed faster than previous assays using hydrazine so that the amount of enzyme used for the assay could be reduced. The recovery of D-3-hydroxybutyrate added to liver samples was 98 +/- 1% (mean +/- SEM, n = 6). The assay was found to be suitable for the measurement of D-3-hydroxybutyrate in samples such as perchloric acid extracts of isolated hepatocytes even when the acetoacetate to D-3-hydroxybutyrate ratio was 4 to 1. This assay presents a reliable alternative to the use of hydrazine and may be used for the assay of D-3-hydroxybutyrate in a variety of physiological and experimental samples.

3-Hydroxybutyric Acid↗
PubMed1983

Analysis of protein-glutathione mixed disulfides by high performance liquid chromatography.

After precipitation of proteins; serum, hepatocytes, or glutathione-derivatized bovine serum albumin, by perchloric acid, dithiothreitol was used to reduce glutathione-protein mixed disulfides in the ether-washed, resuspended pellet. Following neutralization and S-carboxymethylation of free sulfhydral groups in the acid soluble fraction by iodoacetic acid. 2,4-dinitrophenyl derivatives of released compounds were produced by addition of ethanolic fluorodinitrobenzene. The 2,4-dinitrophenyl derivative of S-carboxymethylglutathione was measured by high-performance liquid chromatography. The method was found to be reproducible and limited only by the sensitivity of the glutathione analysis (about 10 pmol/sample). Quantitation of protein-bound glutathione was shown to be independent of the ratio of bound to soluble glutathione as well as the protein concentration in the sample. This method was found to produce glutathione values identical to those measured after borohydride reduction without the problems of foaming, sample loss, and the need of continuous pH adjustment during reduction.

Borohydrides↗
PubMed1983

Quantification without purification of blood and tissue adenosine by radioimmunoassay.

Highly specific anti-adenosine antibodies were produced in rabbits by the injection of N6-carboxymethyl adenosine-methylated serum albumin conjugates. They were used to develop a radioimmunoassay allowing the quantitation of adenosine in the range 0.1-10 pmol per sample. Inosine did not interfere except at 300 times higher concentrations, while AMP (ATP) did not displace the [3H]adenosine tracer even at 10(5) (10(6) ) times higher amounts. Due to the high specificity of the anti-adenosine antibodies, determination of blood and tissue adenosine levels could be performed directly from perchloric acid extracts. Values for human peripheral venous blood from various donors obtained with this procedure varied between 46 and 148 pmol/ml blood. The procedure was also applied to HeLa cultures with low and high intracellular adenosine. The reliability of the method was demonstrated by comparative analyses using HPLC purification of adenosine prior to the radioimmunoassay.

Adenosine↗
PubMed1984

High-performance liquid chromatographic separation and quantification of alanopine and strombine in crude tissue extracts.

A method for the separation and quantification of the levels of alanopine and strombine in neutralized, perchloric acid extracts of tissues of marine invertebrates is presented. The method is based on high-performance liquid chromatographic (HPLC) separation, postcolumn derivatization using o-phthaldialdehyde and sodium hypochlorite, and subsequent fluorometric detection. Isocratic separation results in the rapid elution of alanopine and strombine, with elution times of 4.7 and 5.4 min, respectively. The sensitivity of this method is in the range 50-250 pmol. However, the fluorometric detection approach provides the capability for even greater sensitivity.

Alanine↗
PubMed1984

A fluorometric method for the specific determination of serum arginine with 2,3-naphthalenedicarbaldehyde.

A specific and simple fluorometric method for the determination of serum arginine was developed which consists of (1) deproteinization of serum sample with 10% perchloric acid, (2) blocking thiol compounds with N-ethylmaleimide, (3) fluorescence reaction with 2,3-naphthalenedicarbaldehyde in 0.2 M borate buffer, pH 9.3, in the presence of beta-cyclodextrin (25 min at 20 degrees C, excitation wavelength: 462 nm, emission wavelength: 520 nm). Since the fluorescence reaction is highly specific for arginine, the method requires no other separation step. Linearity, recovery, and reproducibility of the method were found to be satisfactory.

Arginase↗
PubMed1984

Measurement of tissue purine, pyrimidine, and other nucleotides by radial compression high-performance liquid chromatography.

A high-performance liquid-chromatographic (HPLC) method for the rapid separation of purine and pyrimidine nucleotides, NAD+, NADP+, FAD, FMN, UDP-Glc, UDP-glucuronate, and ADP-ribose found in neutralized perchloric acid extracts of rat liver is described. Separation was achieved within 26 min on a radially compressed column of Partisil 10-SAX. The column was eluted with a gradient of sodium phosphate and sodium chloride. The sodium phosphate was purified by passage through tandem columns of anion- and cation-exchange resins to remove uv-absorbing impurities. The sensitivity of this procedure is such that an amount of ATP contained in 10 micrograms of liver can be measured. The recoveries of all nucleotides were between 87 and 107%. In extracts of rat liver interfering substances were found to elute with GDP, and UDP eluted with NADP. Consequently, the tissue contents of UDP and GDP were determined in a second run by measuring the increase in UTP and GTP, respectively, following sample pretreatment with pyruvate kinase (PK). The tissue level of NADP+ was calculated as the difference between the total UDP and NADP+ peak and the increase in UTP following PK treatment. In those nucleotides amenable to enzymatic analysis, namely NAD+, AMP, UDP-Glc, UTP, and ATP, the tissue contents measured enzymatically were not significantly different from those determined by HPLC. However, ADP as measured with PK was found to be 15% higher compared to the HPLC determination.

Animals↗
PubMed1984

A radioenzymatic assay for plasma adenosine.

A method for analysis of plasma adenosine which combines the principles of radioisotope dilution and enzymatic catalysis is presented. Plasma from venous heparinized blood containing the adenosine deaminase inhibitor 2'-deoxycoformycin is mixed with a small amount of [3H]adenosine and extracted with perchloric acid. Using highly purified enzyme and [gamma-32P]GTP as the phosphate donor, the neutralized extract then serves as substrate for adenosine kinase, and the AMP product is purified by high-performance liquid chromatography. Adenosine concentrations in plasma are linearly proportional to 32P/3H ratios in the enzymatically synthesized AMP and are calculated from a standard curve. The advantages of the method are: ease of sample preparation; sensitivity of 20 nM in as little as 0.3 ml plasma; 20 samples per day can be analyzed by a single operator. Care must be used when obtaining plasma since cellular contamination will affect results. Using this assay, human plasma adenosine levels are 0.121 +/- 0.054 microM for males and 0.101 +/- 0.067 microM for females.

Adenosine↗
PubMed1985

Solid-phase purification and analysis of dicarboxylic porphyrins extracted from cultured tumor cells.

We describe here a sensitive method for the purification and analysis of porphyrins present in hematoporphyrin derivative. Hematoporphyrin derivative is a solution containing a complex mixture of dicarboxylic porphyrins such as hematoporphyrin IX, monohydroxyethyl monovinyl deuteroporphyrin isomers, and protoporphyrin IX in addition to porphyrin aggregates of variable molecular sizes. This mixture is known for its ability to be selectively retained by tumor cells and for its cytotoxicity in the presence of light. In order to study the mechanisms of hematoporphyrin derivative uptake and its cellular metabolism, extraction methods are required that combine high recoveries with minimum changes of very labile components. Extraction with perchloric acid: methanol mixtures recovered only some 60% of the porphyrins taken up by tumor cells and artifactual fluorescent spots were seen on thin-layer chromatograms. Improved yields were obtained upon extraction with dimethyl sulfoxide or Triton X-100:4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer mixture, but the extracts were not suitable for reverse-phase thin-layer chromatography (RTLC). The procedure described here consists of extracting porphyrins from cultured tumor cells with a buffered detergent followed by sequential chromatography on DEAE-cellulose columns and on reverse-phase octadecylsilyl cartridges. Identification of the isolated free dicarboxylic porphyrins is conveniently done by RTLC.

Animals↗
PubMed1985

Single-run high-performance liquid chromatography of nucleotides, nucleosides, and major purine bases and its application to different tissue extracts.

A high-performance liquid chromatography (HPLC) method is described for the separation and quantitation of nucleotides, nucleosides, purine bases, and related compounds in one single run. The separation of a standard mixture of at least 24 components is achieved within 35 min on glass columns (30 cm, 3-mm i.d.) with C-18 reversed-phase particles of 5 micron, and ammonium dihydrogen phosphate (0.15 M, pH 6.00) and a slow linear gradient of methanol/acetonitrile (to 15%) as eluting solvent. The method has been applied to microsamples of different cells and tissues. Samples (2.5 mg dry wt) were cooled in liquid nitrogen, lyophilized, and extracted with 0.6 N perchloric acid. After neutralization with potassium bicarbonate, the extract (20 microliter) was directly injected into the column. To illustrate the wide applicability of the method, representative chromatograms are shown of extracts of biopsies from heart tissue, skeletal muscle, and brain and liver and from hepatocytes, erythrocytes, and yeast cells, under different conditions, known to induce changes in purine metabolism.

Animals↗
PubMed1985

Separation and measurement of short-chain coenzyme-A compounds in rat liver by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic method has been developed to measure short-chain CoA compounds in freeze-clamped liver. Seventeen CoA compounds can be quantitated in 37 min using a 3-micron octadecylsilica column (4.6 mm X 7.5 cm). The chromatographic separation of CoA compounds is conducted with a gradient system of sodium phosphate and acetonitrile. The large amount of uv-absorbing, non-CoA material present in liver extracts is eluted earlier than the CoA compounds when the phosphate concentration is 0.2 M. The CoA compounds that can be resolved by this method include acetoacetyl-CoA, acetyl-CoA, butyryl-CoA, CoASH, crotonyl-CoA, dephospho-CoA, glutathione-CoA, 3-hydroxy-3-methylglutaryl-CoA, isobutyryl-CoA, isovaleryl-CoA, malonyl-CoA, 3-methylcrotonyl-CoA, methylmalonyl-CoA, oxidized-CoA, propionyl CoA, succinyl-CoA, and valeryl-CoA. Comparisons at pH 3 and 6 showed that the stability of the CoA compounds is much greater when perchloric acid extracts of rat liver are adjusted to pH 3. Recovery of CoA standards added in tissue extracts ranged from 83 to 107%. The method is linear over the range of 12 to 700 pmol, and this sensitivity allows acetyl-CoA content to be determined in extracts of as little as 0.1 mg of liver. The values for CoA compounds obtained for freeze-clamped liver from starved rats include (units are nmol/g wet weight +/- SE) malonyl-CoA, 1.50 +/- 0.14; glutathione-CoA, 6.57 +/- 1.72; CoASH, 56.06 +/- 2.90; methylmalonyl-CoA, 4.60 +/- 1.27; succinyl-CoA, 13.52 +/- 0.76; 3-hydroxy-3-methylglutaryl-CoA, 7.06 +/- 0.89; and acetyl-CoA, 100.5 +/- 6.4.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl Coenzyme A↗
PubMed1986

Determination of short-chain acyl-coenzyme A esters by high-performance liquid chromatography.

A method for the determination of short-chain acyl-CoA esters in tissue extracts by HPLC has been developed. The acyl-CoA esters were extracted from freeze-clamped rat livers with perchloric acid. The extract was applied to a Sep-Pak C18 cartridge. The cartridge was washed with acidic water, pH 3, followed by petroleum ether, chloroform, and methanol. Then the acyl-CoA esters were eluted from the cartridge with ethanol/water (65:35) containing 0.1 M ammonium acetate. By this procedure, the acyl-CoA esters were concentrated and partially purified. The eluate was analyzed by HPLC using reverse-phase columns of Develosil ODS (0.46 X 15 cm plus 0.46 X 25 cm). The separation of the acyl-CoA esters was conducted with a linear gradient (1.75 to 10%) of acetonitrile. The CoA compounds (malonyl-CoA, succinyl-CoA plus CoASH, methylmalonyl-CoA, 3-hydroxy-3-methylglutaryl-CoA, acetyl-CoA, acetoacetyl-CoA, and propionyl-CoA) were identified and determined by monitoring at 260 nm. Isobutyryl-CoA was used as an internal standard, since the content of this CoA ester was negligible in livers from rats with several metabolic conditions. The lower limit of detection of individual acyl-CoA esters was approximately 50 pmol. Using this analytical method, short-chain acyl-CoA esters were determined in livers from normal and fasted rats.

Acyl Coenzyme A↗
PubMed1986

Simultaneous determination of histamine and N tau-methylhistamine with high-performance liquid chromatography using electrochemical detection.

We have developed a liquid chromatographic method which uses electrochemical detection for the simultaneous quantitation of histamine and N tau-methylhistamine in rat brain. The amines are derivatized with the water-soluble Bolton-Hunter reagent (sulfo B-H). Perchloric acid extracts of rat brains are chromatographed on a strong cation-exchange resin. The eluate is evaporated and allowed to react with sulfo B-H at pH 9.8 at room temperature. The derivatization is complete after 30 s vortexing. The derivatives are purified using a cellulose-phosphate fibrous cation exchanger. They are quantified with an electrochemical detector at a potential of 0.56 V after preoxidizing the sample at 0.47 V. The derivatives of histamine, N tau-methylhistamine, and N alpha-methylhistamine are completely separated without interfering peaks. Since no N alpha-methylhistamine was detected in rat brain it was used as an internal standard. The detection limits are 0.1 pmol of histamine and 0.2 pmol of N tau-methylhistamine. The precision of this method is high, with within-run and between-run coefficients of variation of 2-7% and linearity of 0.999. Both histamine and N tau-methylhistamine peak heights increased significantly and selectively after treatment with pargyline. Because of the high sensitivity, accuracy, and precision, the histamine and N tau-methylhistamine contents of single nuclei of the rat hypothalamus can be routinely quantified.

Animals↗
PubMed1986

Quantitative determination of antiviral nucleoside analog in DNA.

A technique for the analysis of the amount of an antiviral nucleoside analog incorporated into DNA, utilizing enzymatic digestion of DNA, followed by high-performance liquid chromatography is described. The cells or tissue samples were treated with perchloric acid to inactivate the nucleases, then digested with pronase in the presence of EDTA. DNA was purified by CsCl centrifugation followed by Sephadex chromatography and treatment with deoxyribonuclease 1 and venom phosphodiesterase. The deoxyribonucleoside monophosphates and the monophosphate of the nucleoside analog liberated from DNA were separated and quantitated by HPLC analysis and measurement of radioactivity. This assay is more sensitive, specific, and precise than the determination of DNA density shift. It is also applicable for nucleoside analogs which do not change the density of DNA either because of their structure or their very small degree of incorporation.

Animals↗
PubMed1986

Automated microanalysis of adenosine phosphates, phosphocreatine, creatine, and lactate in muscle.

An automated enzymatic procedure suitable for determination of ATP, ADP, AMP, phosphocreatine, creatine, and lactate in needle biopsies of human skeletal muscle (ca. 30 mg dry wt) using a fast centrifugal analyzer (Multistat III, Instrumentation Laboratory Inc.) is presented. Coefficients of variation ranged from 0.7 to 4.2% for multiple determinations of ATP, ADP, phosphocreatine, and creatine; from 6 to 24% for lactate; and from 9 to 20% for AMP. The procedure should be usable, with appropriate modification, with other tissues and with other fast centrifugal analyzers. Muscle samples are collected into liquid freon, lyophilized, and extracted with 600 microliter of 0.65 M perchloric acid. Neutralized supernatants can be stored for up to 3 years at -80 degrees C with no significant deterioration. The procedure takes much less time than similar manual procedures and gives better reproducibility, particularly for ADP and AMP.

Adenine Nucleotides↗
PubMed1987

High-performance liquid chromatographic assays for bufuralol 1'-hydroxylase, debrisoquine 4-hydroxylase, and dextromethorphan O-demethylase in microsomes and purified cytochrome P-450 isozymes of human liver.

Bufuralol, debrisoquine, and dextromethorphan are three prototype substrates of the common genetic deficiency of oxidative drug metabolism in man known as debrisoquine/sparteine-type polymorphism. We describe assays for the in vitro metabolism of (+)- and (-)-bufuralol, debrisoquine, and dextromethorphan in human liver microsomes and reconstituted purified cytochrome P-450 isozymes. These assays combine nonextractive sample preparation by precipitation of protein with perchloric acid with reversed-phase inorganic ion-pair HPLC and fluorescence detection. The minimal detectable levels of the major metabolites formed are 1'-hydroxybufuralol, 0.1 ng/ml; 4-hydroxydebrisoquine, 0.8 ng/ml; and dextrorphan, 0.1 ng/ml. Formation of these metabolites is linear for at least 45 min and between 1 and 100 micrograms of microsomal protein. Comparative kinetic analysis of the three monooxygenase reactions in human liver microsomes revealed an apparent biphasicity of (+)- and (-)-bufuralol 1'-hydroxylation and dextromethorphan O-demethylation but monophasic formation of 4-hydroxydebrisoquine in the substrate concentration range (less than 1 mM) studied. These data, in combination with those obtained by purified human cytochrome P-450 isozymes indicate the involvement of the same enzyme in the metabolism of all three substrates investigated. However, additional and distinct activities contribute to the metabolism of (+)- and (-)-bufuralol and dextromethorphan.

Chromatography, High Pressure Liquid↗